FIG. 8.

KSHV trafficking in HMVEC-d cells and association with early and late endosomes. (A) Uptake of transferrin. Uninfected cells were pulsed with Alexa Fluor 594-tagged transferrin (25 μg/ml) for 5 min or 30 min at room temperature, fixed, permeabilized, washed in PBS, blocked with 5% BSA, and incubated with anti-mouse EEA-1 antibodies for 1 h at room temperature. The cells were washed, reacted with secondary antibodies, washed, mounted in antifade reagent with DAPI, and visualized under a fluorescence microscope. Red arrows indicate colocalization of transferrin with EEA-1. Magnification, ×40. (B and C) HMVEC-d cells grown in eight-chamber slides were infected with KSHV at an MOI of 10 for the indicated times, fixed, permeabilized, and stained with mouse anti-EEA-1 or anti-LAMP-1 and rabbit anti-ORF65 antibodies for 1 h at room temperature. Cells were washed, incubated with anti-mouse Alexa Fluor 488 (green) and anti-rabbit Alexa Fluor 594 (red) antibodies for 30 min at room temperature, washed, mounted in DAPI, and visualized. Red arrows indicate colocalization of KSHV capsid and LAMP-1. Magnification, ×40. Boxed areas are shown enlarged in the right-hand panels. ', min; UN, uninfected.